Matrix coating for sensing surfaces capable of selective biomolecular interactions, to be used in biosensor systems

ABSTRACT

A matrix coating suitable for use in a biosensor is provided. This matrix coating comprises a hydrogel bound to a surface and via which a desired ligand can be bound. This hydrogel is activated to contain charged groups for bringing about the concentration of biomolecules carrying an opposite charge to that of said charged groups, and reactive groups for covalently binding the biomolecules concentrated to the matrix coating.

This application is a continuation of application Ser. No. 08/058,265filed on May 10, 1993, now abandoned, which is a continuationapplication of Ser. No. 07/681,531, filed as PCT/SE89/00642 Nov. 9,1989, now U.S. Pat. No. 5,242,828 issued Sep. 7, 1993.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of biosensors and is morespecifically concerned with methods for providing metal surfaces withsurface layers capable of selective biomolecular interactions. Thepresent invention also comprises activated surfaces for coupling adesired ligand; surfaces containing bound ligand; and the use of suchsurfaces in biosensors.

2. Description of the Related Art

According to Aizawa (1983) a biosensor is defined as being a uniquecombination of a receptor for molecular recognition, for example aselective layer with immobilized antibodies, and a transducer fortransmitting the interaction information to processable signals. Onegroup of such biosensors will detect the change which is caused in theoptical properties of a surface layer due to the interaction of thereceptor with the surrounding medium. Among such techniques may bementioned especially ellipsometry and surface plasmon resonance. Inorder for these types of techniques to work satisfactorily in actualpractice certain requirements have to be fulfilled--i.e., therequirement that the sensing surface (or measuring surface) employed caneasily be derivatized so that it will then contain the desired receptor,and moreover that it will not produce any (or only negligible)non-specific binding, i.e., binding of components other than those thatare intended. In somewhat simplified terms the technique of surfaceplasmon resonance--by abbreviation SPR, as derived from the initialssurface plasmon resonance may be said to be a technique in which changesin the refractive index in a layer close to a thin metal film aredetected by consequential changes in the intensity of a reflected lightbeam (see for example Raether, H (1977)).

The sensing surface is composed of receptors or "ligands" as they willbe called henceforth, these being generally molecules or molecularstructures which interact selectively with one or more biomolecules.

The metal film is applied on a substrate of a type that is suitable forthe measuring method employed. In the case of SPR, this means that adielectric material, e.g., in the form of a glass plate, is used fordirecting a light beam to the metal surface.

According to most of the publications that have come forth up to now,SPR procedures when applied to detecting biomolecules have been carriedout simply by adsorbing the biomolecule in question directly to themetal surface and then studying the consequential effect on themeasuring signal. In a next step, this surface could optionally be usedfor binding a new layer of molecules (ligands) having an affinity forthe first-bound layer of molecules. Thus for instance Liedberg, B. etal., (1983), in a first work indicating the potential of SPR technologyfor biochemical analyses, adsorbed at first a monolayer of IgG to asilver surface and then adsorbed an anti-IgG layer to said monolayer, inorder to then study the effect with respect to the resultant change inthe resonance angle.

Others too, e.g., Cullen D. C. et al., (1987/88), have utilizedadsorption of biomolecules directly to a metal surface when studyingimmune complex formation in the IgG/anti-IgG system using the SPRtechnique with a gold-coated diffraction grating.

EP 257955 describes a method according to which the metal film is coatedwith silica and optionally treated with a silanizing reagent; and in EP202021 the metal film has been coated with an organic layer that maycontain for example an antibody to a specific antigen. Although thepossibility of the antibody being bound covalently is indeed mentionedin that specification the actual nature of the organic layer is notdisclosed or indicated at all, and the same applies to the manner inwhich the organic layer is produced.

According to EP 254575 an optical structure of the type such as issuitable for e.g. SPR applications may be produced by coating the metalfilm with a layer of an organic polymer, by means of the so-called"solvent casting technique". In a preferred embodiment cellulose nitrateis employed, and a number of well-known methods are mentioned forbinding biospecific ligands to the layer.

Publications of this kind, while giving indications of the potential ofthe method, also demonstrate some of the limitations inherent in thetechnical solutions proposed.

As pointed out in, for instance, EP 254575, one of the problems is thatbiomolecules may be subject to an at least partial inactivation due todirect contact with metallic and certain inorganic surfaces. Anothercomplication is that some ligands which may be desirable for somespecial applications cannot be adsorbed in a stable manner to a metalsurface and thus cannot be expected to give reproducible results. Stillanother problem is that many of the media occurring in biochemicalsystems have a corrosive effect on the metal surface.

Although problems of these kinds may be solved at least in part by wayof a process according to EP 254575, a construction of this type has anumber of obvious drawbacks. A polymeric coating in the form ofcellulose nitrate--as according to a preferred embodiment--will put alimit on the number of possible applications inasmuch as it is awell-known fact that biomolecules can be adsorbed irreversibly tocellulose nitrate films. In biosensor systems based optical surfacedetection technology such a phenomenon may give rise to ambiguous andnon-reproducible signals due to non-specific interaction between thesensing surface and components present in, for instance, human serumsamples. Such side effects have been compensated for in EP 254575 byusing a combination of a measuring and a reference surface. Arequirement for the working of this method is that the nonspecificcontribution is equally great on both surfaces; but this condition isnot always fulfilled in actual practice.

Another problem is pointed out in EP 226470 regarding the production ofconstructs similar to the one mentioned above (see also U.S. Pat. No.4,415,666). From the specification it can be seen how difficult it is toobtain acceptable stability, uniformity and reproducibility of thepolymeric coating; and the consequential negative effects in cases wherebiosensor systems are employed will be readily appreciated.

Although for many practical uses a polymeric coating of the cellulosenitrate type having a thickness of 15-20 nm may indeed providesufficient protection from corrosion, there is nevertheless an obviousrisk that smaller molecules may penetrate through such a layer and causean irreversible change in the metal surface. As shown below, sulfurcompounds such as will be encountered in some situations associated withthe present type of measurements--e.g., in cases where organic thiolcompounds are used for reducing disulfide bonds in proteins--have a highaffinity for noble metals and upon being adsorbed will produce anuncontrolled alteration of the optical properties of the metals. It hasalso been shown that a polymeric coating of the cellulose nitrate typemay be damaged by, e.g., detergent treatment with 2% SDS (see EP254575).

SUMMARY OF THE INVENTION

A generally useful sensing surface for biosensor systems, especiallySPR, should fulfil the following desiderata:

It should be chemically resistant to the media employed.

It should be compatible with proteins and other biomolecules and shouldnot interact with any molecules other than those desired.

It should be capable of providing for covalent binding of such a largenumber of ligands as is required for a general applicability of thistechnique to a variety of analytical problems.

For the sake of obtaining a high degree of sensitivity and dynamics, thesurface should provide a tridimensional matrix for the sample solutionfor binding the target molecules therein. In this manner a greater partof the volume influencing the resonance effect, by way of its refractiveindex, will be utilized as compared to cases where a two-dimensionalsurface would be used.

We have now constructed a surface which in its preferred embodiment willfulfill all of these desiderata quite well.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will be better understood from the following detaileddescriptions taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention, in which:

FIG. 1 shows the response curve obtained with (1) injection of culturemedium, (2) injection of culture medium containing monoclonal antibody,and (3) regeneration with 0.1M glycine, pH 2.5, upon injection aftercovalent immobilization of rabbit anti-mouse light chain (RAMLC)antibodies onto the sensing surface described in Example II.3.

FIG. 2 shows the standard curve for various concentrations of amonoclonal IgG1 antibody. Within the range of 5-100 μg antibody per ml,the accuracy of the dose-response curve is better than ±10%.

FIG. 3 shows sequential injections of subclass-specific reagents andidentification of bound monoclonal as an IgG1 antibody: (1) Monoclonalantibody binds to the surface, this being followed by injections of (2)anti-IgG2a, (3) anti-IgG3, (4) anti-IgG2b, and (5) anti-IgG1 (whichbinds to the surface).

DETAILED DESCRIPTION OF THE INVENTION

The followed detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the following detailed description should not be construed to undulylimit the present invention, as modifications and variations in theembodiments herein discussed may be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

The metal surface is constituted by a film of a free electron metal suchas e.g. copper, silver, aluminum or gold. These metals give differentresonance effects, and although silver is very good in this respect wehave nevertheless decided--in view of corrosion stabilityconsiderations--that gold should be the preferred metal. For binding thedesired biospecific ligand we have applied a monolayer of an organicmolecule X-R-Y to the metal surface. This monolayer is densely packed onthe surface, and in addition to being used for binding the ligand itforms an efficient barrier layer which is very stable upon storage andprotects the metal surface from chemical corrosion.

Generally, modification of gold surfaces with certain types of sulfurcompounds has been described by, for example, Nuzzo R. G. et al. (1983),Porter M. D. et al. (1987), and Troughton E. B. et al. (1988).

X-R-Y in the form of a densely packed monolayer becomes attached to themetal in conformity with the principles described in the above-citedpublications, the bonds being partially covalent in character; X bindsto the metal and Y serves for coupling with functional ligands. This maybe effected either in that the ligand is coupled directly to Y,optionally after activation of Y, or in a biocompatible porous matrixlike, for instance a hydrogel, is bound to the barrier layer via Ywhereupon this matrix is utilized for binding the ligand.

X belongs to one of the following groups:

asymmetrical or symmetrical disulfide (--SSR'Y', --SSRY), sulfide(--SR'Y', --SRY), diselenide (--SeSeR'Y, --SeSeRY) selenide (--SeR'Y',--SeRY)

thiol (--SH), nitrile (--CN), isonitrile, nitro (--NO₂), selenol (--SeH), trivalent phosphorous compounds, isothiocyanate, xanthate,thiocarbamate, phosphine,

thioacid or dithioacid (--COSH, --CSSH).

R (and R') is (are) a hydrocarbon chain which may optionally beinterrupted by hetero atoms and which is preferably straight(=non-branched) for the sake of optimum dense packing, and containsoptionally double and/or triple bonds. The length of the chain exceeds10 atoms. Shorter chains will yield layers of poorer stability. Chainlengths of 12-30 atoms are currently preferred. The carbon chain mayoptionally be perfluorinated.

Y and Y', which are preferably the same, have properties such that theycan bind the target substance directly or after activation. Y (and Y')may thus be any among the great number of groups which are used forimmobilization in liquid chromatography techniques, for instance ahydroxyl, carboxyl, amino, aldehyde, hydrazide, carbonyl, epoxy or vinylgroup. There are many articles to be found in the literature dealingwith coupling of various ligands such as, e.g., biomolecules, with theaid of these or other groups; available alternatives of choice willtherefore be readily obvious to persons skilled in the art.

An obvious variant of this concept involves adsorbing a mixture ofdifferent organic molecules X-R-Y. This may be done for the purpose ofobtaining an increased capacity for further derivatization or for thepurpose of obtaining multifunctional surfaces. Furthermore, the barrierlayer may be formed by means of various types of more complex moleculessuch as, for instance, molecules containing two or more carbon chainslinked to each other in a manner as described by Regen S. L. et al.(1986).

By crosslinking the molecules in the barrier layer, its stability couldbe further increased, if necessary. This could be achieved viafunctional groups in R or Y, for instance by photoinitiatedpolymerization of double or triple bonds in R; see for instanceRingsdorf H. et al (1988 ).

If the desired ligand or biomolecule is bound directly via Y to thebarrier layer several among the above-mentioned desiderata have beenfulfilled, and acceptable results are obtainable in at least somepractical application instances. According to a preferred embodiment,however, a biocompatible porous matrix like, for instance, a hydrogel,is coupled to the barrier layer, and this matrix, which has a thicknessof from a few angstroms to several thousand angstroms, is employed forimmobilizing a ligand that is suitable for the target biomolecule. Inactual practice the thickness of the matrix layer is chosen so as tosuit the dimension of the measuring signal in the measuring space of themeasuring system employed, to thus create an optimum set of detectionconditions. In SPR applications, the thickness of the matrix layer ispreferably 5 to 10,000 angstroms, especially 5 to 1,000 angstroms. Aconsiderably higher ligand density per area unit is obtained in a matrixas described here, compared to the prior art technique, in which bindingof molecules occurs mainly in monolayers, thus giving a considerablyenhanced measuring signal, making the system useful in a larger dynamicrange.

The hydrogel in the form as contemplated here and which at present isthe preferred embodiment of the matrix, may be defined as according toMerrill et al. (1986). Hydrogel coupling is essential for obtaining asensing surface fulfilling above all the aforesaid desiderata withrespect to protein compatibility and minimized nonspecific interaction.Merrill et al. have described a large number of examples showing suchproperties in the hydrogel surfaces. Depending on t2he actual practicalapplication contemplated, any particular hydrogel may be chosen fromamong several available alternatives of choice.

The hydrogel may be, for example a polysaccharide such as agarose,dextran, carrageenan, alginic acid, starch, cellulose, or derivatives ofthese such as, e.g., carboxymethyl derivatives, or a water-swellableorganic polymer such as, e.g., polyvinyl alcohol, polyacrylic acid,polyacrylamide, polyethylene glycol. The hydrogel can be derivatized tocontain hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy, or vinylgroups for immobilizing the desired ligand, and optionally, abiospecific ligand bound via said groups. The hydrogel can contain a2-aminoethanethiol derivative.

In particular, polysaccharides of the dextran type which arenon-crystalline in character, in contrast to e.g., cellulose, are verysuitable in these contexts. Dextran has been used to a very great extentin chromatographic procedures as a matrix for the binding ofbiomolecules; one of the advantages inherent in the present concept isactually that this entire technology is now available also for biosensorapplications, viz., for the final step of the biosensor technique inwhich a suitable ligand is being immobilized. The hydrogel may either bebound to the barrier layer "metal-X-R-Y" or may be generated in situfrom a suitable solution of monomeric material. Further alternativetreating steps such as, for example, subsequent crosslinking of thehydrogel, will naturally be readily obvious to persons skilled in theart.

This type of surface modification can be utilized also in other fieldsof technology where a specific, or alternatively, a low non-specific,interaction is required between a surface on one hand and proteins orother biomolecules on the other hand. Examples that may be mentioned areparts of chromatographic systems for biomolecule separations where,e.g., metal filters may have their surface modified in a manner asindicated above. It would also be possible to construct capillary-typechromatographic columns in conformity with these principles.Furthermore, it is evident that a surface structure may be modified soas to acquire biocompatibility, for use in environments of the "in vivo"type. Depending on the particular field of use contemplated, the actualchoice of, for example, the hydrogel, can be made such that undesiredinteractions are minimized. To those skilled in the art, a number ofadditional fields of use will be readily obvious, along the lines of theaforesaid examples.

According to an embodiment further illustrating the invention, a layerof 16-mercaptohexadecanol is bound to the gold film whereupon thehydroxyl groups on the barrier layer are epoxy-activated by means ofbeing treated with epichlorohydrin. In a subsequent step, dextran isattached to the barrier layer via ether linkages. The dextran matrix isnext activated for binding ligands according to known techniques, forexample in accordance with one of the following principles.

In one embodiment, a hydrazide function is created in the dextran matrixfor binding ligands containing aldehyde groups, for example antibodiesin which the carbohydrate chain has been oxidized so that it thencontains an aldehyde function. In this instance, the dextran matrix isinitially modified with carboxymethyl groups which are partly reacted toform hydrazide groups. With this activated matrix at least two importantadvantages are obtained: 1) This matrix contains unreacted carboxylgroups which in low ionic strength conditions will act as ionexchangers, and by electrostatic interaction the ligand which is to beimmobilized is concentrated to the dextran matrix. 2) This matrix willvery efficiently bind the ligand thus concentrated at the surface, viz.by condensation of ligand aldehyde groups with the hydrazide function ofthe matrix.

According to another embodiment, a part of the carboxyl groups incarboxymethyl-modified dextran are modified so as to give reactive esterfunctions, e.g., by treatment with an aqueous solution ofN-hydroxysuccinimide and N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimidehydrochloride. In the same way as in the case described above, theresidual charges id est unreacted carboxyl groups will contribute toeffecting a concentration of ligands on the surface. Ligands containingamine groups such as, for example, proteins and peptides, may then becoupled to the dextran matrix by covalent bonds.

According to an alternative procedure, the aforesaid reactive ester isutilized for reaction with a disulfide-containing compound such as forinstance 2-(2-pyridinyldithio) ethanamine; in this manner a matrix isobtained which contains disulfide groups, and these can be employed forcoupling thiol-containing ligands such as, for example, reduced F(ab)fragments of immunoglobulins (see Brocklehurst K et al (1973)). Aftercleavage of the disulfide bonds, for instance by reduction orthioldisulfide exchange, the thiol modified surface formed can be usedfor coupling of a disulfide-containing ligand such as, for instance,N-succinimidyl 3-(2-pyridinyldithio) propionate (SPDP) modifiedproteins.

The advantage of this procedure is that the ligands via, for example, areduction step can be cleaved off to give a sensing surface withreactive thiols. This thiol-modified surface can in an analogousprocedure be used for renewed covalent coupling of thiol- ordisulfide-containing ligands. In this way the capability of chemicalregeneration of the sensing surface can be obtained, which can be usedfor general utilization of the same surface for couplings of severaldifferent ligands. The procedure can also be used when, for example, abiological interaction is studied, and this interaction cannot be brokenwhile retaining biological activity of the immobilized ligand.

One important aspect of the present invention is that one or more of thelayers forming the sensing surface to be used in a given analysis can besynthesized and/or functionalized in situ by adding the appropriatereagents to the surface in a flow-through cell in a biosensor system.

To sum up, there are a multitude of ligands that can be employed for thedetection of biomolecules by means of interacting therewith. It will bereadily evident that ion exchanging groups, metal chelating groups andvarious types of receptors for biological molecules--such as are knownfrom conventional liquid chromatographic procedures--may be employed forthe construction of systems which are suitable for selection purposeseven in complex measuring systems.

This novel type of sensing surfaces permits measurements to be carriedout in systems comprising multiple sensing surfaces for analysing aplurality of sample components; in such a system a very high degree ofversatility is obtained if each particular sensing surface isfunctionalized in situ so as to acquire its desired specificity. In thiscase, such a sensing surface initially contains an activated dextranlayer; functionalization is effected by means of passing the appropriateligand solutions over each respective one of the sensing surfaces.Thereafter the sample solutions are passed over the multipe sensingsurfaces, and bound components are detected.

A sensor unit having at least two sensing surfaces, as well as a methodfor its functionalization, is the object of copending PCT applicationentitled "Sensor unit and its use in biosensor systems" (based uponSwedish patent application No. 8804074-6), the disclosure of which isincorporated by reference herein.

In one embodiment, so-called chimaeric molecules (bi- or polyfunctionalmolecules) are used for functionalizing the sensing surfaces. Thechimaeric molecules comprise one part that will bind to the basalsurface, for example to the aforesaid dextran-coated sensing surface,and one part having an affinity for the biomolecule to be detected. Inthe case of dextran, the chimaeric molecule may consist of an antibodyto dextran which is conjugated to a biospecific ligand, e.g., animmunoglobulin. With a series of such chimaeric molecules, which thuscontain a dextran antibody and a group of a different specificity, aso-called measuring cassette containing several sensing surfaces of thesame type in one instrument may be activated in a simple manner forparallel detection of a plurality of biomolecules. According to analternative process, a sensing surface is modified with a so-calledhapten for binding chimaeric molecules to the surface. For example, areactive ester surface as described above may be derivatized with atheophylline analogue which is then employed for binding chimaericmolecules. In this case the chimaeric molecule consists of an antibodydirected against theophylline and conjugated with a biospecific ligand.In light of these embodiments, it will be eminently clear that a highdegree of versatility is attainable when surfaces according to thepresent invention are used, inasmuch as the users can employ identicalbasal surfaces for attaching thereto any of their desired ligands (= thedesired receptor) by way of a simple procedure.

The invention relating to (i) the aforesaid methods for providing metalsurfaces with surface layers capable of selective biomolecularinteractions, to be used in biosensor systems, (ii) the above-describedsurfaces, and (iii) their use in biosensors will now be illustrated bymeans of the following examples, which are non-limitative. The metal, ofcourse is applied to a substrate that is suitable for the particularmeasuring method contemplated in each instance; e.g., a glass plate inthe case of SPR. In view of the fact that the selection of thesubstrates does not form part of this invention, the followingexemplification as well as the present specification as a whole dealsonly with the sensing surface as such, id est the free metal surfacewith the attached layers thereon.

The sensing surfaces of the invention can be used in various biosensorsystems and especially in SPR, e.g., of the type described in copendingPCT application entitled "Optical Biosensor System" (based upon Swedishpatent application No. 8804075-3), the disclosure of which isincorporated by reference herein.

I. Examples of producing the sensing surface

I.1 Synthesis of 16-mercaptohexadecanol

16-mercaptohexadecanol was synthesized in accordance with the followingreaction scheme: ##STR1##

The 16-mercaptohexadecanoic acid methyl ester (IV) was preparedaccording to well-known methods (Crossland R. K. et al. (1970), Ghosh S.S. et al. (1987) and Volante R. P. (1981), plus references cited inthese publications).

Reduction of (IV) to 16-mercaptohexadecanol was carried out as follows:

16-mercaptohexadecanoic acid methyl ester, 12.0 g (41.7 mmol) dissolvedin 70 ml of toluene was added cautiously, dropwise and with vigorousstirring, to 70 ml (70 mmol) of lithium aluminumhydride-bis-tetrahydrofuran (1M) in toluene. During this addition thetemperature was maintained below 25° C. The reaction was allowed toproceed at room temperature for 20 minutes. Excess hydride wasdecomposed by means of ethyl acetate followed by 100 ml of 2Mhydrochloric acid. The layers were separated. The aqueous layer wasextracted with 100 ml of toluene. The combined organic layers werewashed with 100 ml of 2M sodium hydrogen carbonate, dried with magnesiumsulfate and evaporated.

Yield: 10.0 g (87.2%); purity 96% according to GLC.

The product was purified by repeated recrystallization from methanol. Apurity of >99% has been considered acceptable. Melting pt 55.0-56.0° C.

I.2 Basic coupling of Au-coated glass surfaces

I.2.1 Chemisorption of 16-mercaptohexadecanol

A 5" gold-coated glass wafer was placed into a petri dish (i.d. 16 cm)provided with a cover. 40 ml of a 5.0 mM solution of16-mercaptohexadecanol in ethanol/water 80/20 were poured over thesurface. The petri dish was incubated on a shaker incubator at 40° C.for 20 minutes. The surface was washed with 5×50 ml ethanol, 50 mlethanol/water 80/20, and 5×50 ml water. Cyclic voltametric analysis ofthe surface showed the film to effectively prevent oxidation of thegold.

I.2.2 Treatment with epichlorohydrin

The surface coated with 16-mercaptohexadecanol was contacted with asolution of 2.0 ml of epichlorohydrin in 20 ml of 0.4M sodium hydroxideand 20 ml of diethylene glycol dimethyl ether. Reaction was allowed toproceed in a shaker incubator at 25° C. for 4 hours. The surface waswashed with 3×50 ml water, 2×50 ml ethanol, and 5×50 ml water.

I.2.3 Treatment with dextran

13.5 g of dextran (T500, Pharmacia) were dissolved in 40.5 ml of water.4.5 ml of 1M sodium hydroxide were added and the solution was pouredover an epichlorohydrin-treated surface. This was followed by 20 hoursof incubation in a shaker incubator at 25° C. The surface was washedwith 15×50 ml of 50° C. water.

I.3 Derivatization of basal surfaces

I.3.1 Synthesis of a hydrazide surface

Bromoacetic acid, 3.5 g, was dissolved in 27 g of 2M sodium hydroxidesolution. The mixture was poured over a dextran-treated surfaceaccording to 1.2.3 and incubated in a shaker incubator at 25° C. for 16hours. The surface was washed with water, whereupon the aforesaidprocedure was repeated once.

After having been washed, the surface was given a 5 min. treatment with0.8 g of N-(3,dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride(EDC) in 20 ml of water, followed by addition of 4.0 ml of hydrazinehydroxide in 20 ml of water. The surface was incubated in a shakerincubator at 25° C. for 16 hours and then washed with water.

I.3.2 Synthesis of a surface having a reactive ester function

0.69 g of N-hydroxysuccinimide and 1.15 g of EDC were dissolved in 30 mlof water. The mixture was poured over a carboxymethyl-modified dextransurface according to I.3.1 and incubated in a shaker incubator at 25° C.for 60 minutes. The surface was washed with water.

I.3.3 Synthesis of theophylline surface

A solution of 5 mM 8-(3-aminopropyl)-theophylline (R. C. Boguslaski etal., 1980) in 0.1M carbonate buffer, pH 8.0, was incubated with anN-hydroxysuccinimide ester activated dextran surface (according toExample I.3.2) overnight at 25° C., whereupon the surface was washedwith water.

II Coupling of ligands to derivatized basal surfaces

II.1 Anti-IgE antibody

Anti-IgE antibody (Pharmacia Diagnostics AB) in 10 mM acetate buffer, pH5.5, was oxidized with 10 mM sodium periodate for 20 minutes on an icebath according to the method described by O'Shannessy (1985). Afterreplacement of the buffer, the antibody was coupled to thehydrazide-modified dextran surface (Example I. 3) in 10 mM acetatebuffer, pH 4.0. Antibody that had not been bound was eluted with 0.1Mglycine, pH 2.5.

II.2 Antibeta-2-microglobulin antibody

Antibeta-2-microglobulin antibody (Pharmacia Diagnostics AB) wasoxidized and coupled as in Example II.1 to the hydrazide-modifieddextran surface.

II.3 Rabbit anti-mouse light chain antibody (RAMLC)

RAMLC antibody in 10 mM acetate buffer, pH 5.5 (Pharmacia DiagnosticsAB) was coupled for 20 minutes to an N-hydroxysuccinimide esterderivatized dextran surface (according to Example I.3.2), whereuponunbound antibody was washed off by rinsing of the surface in PBS buffer,pH 7.4, and in 0.1M glycine, pH 2.5.

III Biomolecule assays using the SPR technique

The sensing surface was introduced into an SPR measuring device with aflow cell. After adjustment of the optical instrumentation, themeasurement signal was studied as a function of time under constant flowconditions.

III.1 Determinations of concentrations and subclass identities ofmonoclonal antibodies

Culture medium with monoclonal antibodies was injected after covalentimmobilization of RAMLC antibodies onto the sensing surface (ExampleII.3). FIG. 1 shows the response curve obtained with (1) injection ofculture medium, (2) injection of culture medium containing monoclonalantibody, and (3) regeneration with 0.1M glycine pH 2.5. FIG. 2 showsthe standard curve for various concentrations of a monoclonal IgG1antibody. Within the range of 5-100 μg antibody per ml the accuracy ofthe dose-response curve is better than ±10%. FIG. 3 shows sequentialinjections of subclass-specific reagents and identification of boundmonoclonal as an IgG1 antibody: (1) Monoclonal antibody binds to thesurface, this being followed by injections of (2) anti-IgG2a, (3)anti-IgG3, (4) anti-IgG2b, and (5) anti-IgG1 (which binds to thesurface). To verify that the system is reversible and repeatable, theantibody binding and subclass identification have been repeated 100times on the same surface.

III.2 Affinity studies and kinetic studies with anti-theophyllineconjugates as carriers of sensor molecules

Protein A and protein G were introduced to a theophylline surface in theform of conjugates with an anti-theophylline antibody (chimaericmolecules). In this way we could study the interactions between proteinA or protein G and monoclonal antibodies of different subclasses.

Preparation of conjugates

Monoclonal anti-theophylline antibody No. 459, monoclonal antibodies ofvarious different IgG subclasses, monoclonal anti-IgE antibodies of IgG1subclass No. E164, 95 and 121 and IgE were obtained from PharmaciaDiagnostics AB. The anti-theophylline antibody was digested with pepsinto form F(ab)'-2 fragments or was employed as the intact antibody.Protein A, protein G and SPDP were obtained from Pharmacia LKBBiotechnology AB.

Protein A--anti-theophylline conjugate was prepared by means of SPDPmodification of both of these molecules in accordance with the methoddescribed by Carlsson et al. (1978). After reduction of modified proteinA with 10 mM DTE (1,4-dithioerythritol), 3.8 mg of anti-theophyllinehaving a modification degree of 1.8 (pyridyldisulfide groups permolecule) were mixed with 13.8 mg of reduced protein A having amodification degree of 1.3 (thiols/molecule).

Conjugation was allowed to proceed overnight in 0.1M phosphate bufferwith 0.1M sodium chloride at pH 7.7. Conjugates of protein G withF(ab)'-2 fragments of anti-theophylline were prepared in an analogousmanner.

Analysis

Sensing surfaces with bound theophylline are readily functionalizablewith the above-described conjugates. With two parallel sensing surfaces,one of them functionalized with the protein A conjugate and the otherwith the protein G conjugate, it was possible to very quickly comparethe affinities of protein A and protein G, respectively, for a series ofimmunoglobulins. The results obtained confirmed the differences in theserespects as reported by, e.g., Guss et al. (1986).

This experiment demonstrates not only the possibility of rapidlycarrying out qualitative measurements of kinetics and affinities butalso the flexibility of the techniques employing a sensing surfaceaccording to the invention, inasmuch as, with the appropriate reagent ineach respective case, such a sensing surface is utilizable for a widerange of different assays.

III.3 Assay for beta-2-microglobulin using the so-called sandwichprocedure

Measurement of beta-2-microglobulin (Pharmacia Diagnostics AB) wascarried out with a sensing surface containing anti-beta-2-microglobulinantibodies according to Example II.2. The measuring signal for thebeta-2-microglobulin binding to the sensing surface was recorded as afunction of its concentration in solution, both directly (primaryresponse) and after signal enhancement by means of a secondaryimmunosorbent-purified antibody which binds to the surface via theprimarily bound beta-2-microglobulin to thus form a so-called sandwichstructure (secondary response). This obvious and experimentally simpleprocedure provides an at least 10 times lower detection level.

IV Determination of the amount of adsorbed protein to sensing surfaces

The quantification of the amount of proteins adsorbed to thecarboxymethyl modified dextran surface described in I.3.1 was conducted,using a radioactive method. Thus, a ¹⁴ C- or ³⁵ S-labelled protein(immunoglobulin G, chymotrypsinogen A and transferrin) was concentratedby ion-exchange to a surface placed in an SPR measuring device. Thechange in angle was measured whereafter the surface was dried andremoved from the SPR measuring device. The amount of ¹⁴ C-labelledprotein on the surface was then quantified in a device which measuredthe beta-radiation from the surface. Calibration of the radioactivemethod gave the absolute value of the surface concentration of adsorbedprotein. FIG. 4 shows the correlation of the response from the SPRmeasuring device with the surface concentration obtained from thebeta-radiation measurement, for 78 determinations made under variousexperimental conditions. Surfaces with protein concentrations up to 50ng/mm² were obtained, corresponding to approximately 10 dense monolayersof transferrin. This shows the capacity of the described hydrogel anddemonstrates the great measuring signal enhancing effect achieved byusing a sensing surface with a biocompatible porous matrix according tothe present invention. Similar surface concentrations were reached byimmobilizing proteins to surfaces with reactive ester functions, whichalso points out the possibility of making "multilayer-couplings" ofproteins for enhanced dynamics in various SPR measurements.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

References

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We claim:
 1. A matrix coating suitable for use in a biosensor,comprising a hydrogel which is bound to a surface and via which adesired ligand can be bound, which hydrogel is activated to contain (i)charged groups for bringing about a concentration of biomoleculescarrying an opposite charge to that of said charged groups, and (ii)reactive groups for covalently binding said biomolecules concentrated tosaid matrix coating.
 2. The matrix coating according to claim 1, whereinsaid hydrogel is a polysaccharide or a swellable organic polymer.
 3. Thematrix coating according to claim 2, wherein said hydrogel is apolysaccharide selected from the group consisting of agarose, dextran,carrageenan, alginic acid, starch, and cellulose, and a derivative ofany of the foregoing.
 4. The matrix coating according to claim 3,wherein said hydrogel consists of dextran.
 5. The matrix coatingaccording to claim 4, wherein said charged groups and said reactivegroups of said dextran are carboxyl groups, part of which are in theform of reactive esters, hydrazides, thiols, or reactivedisulfide-containing derivatives.
 6. The matrix coating according toclaim 5, wherein the reactive disulfide-containing derivative is theproduct of a reaction between a reactive ester and 2-(2-pyridinyldithio)ethanamine.
 7. The matrix coating according to claim 5, wherein saidhydrogel contains a 2-aminoethanethiol derivative.
 8. The matrix coatingaccording to claim 2, wherein said hydrogel is a swellable organicpolymer selected from the group consisting of polyvinyl alcohol,polyacrylic acid, polyethylene glycol, and polyacryl amide.
 9. Thematrix coating according to claim 2, wherein said charged groups andsaid reactive groups of said hydrogel are carboxyl groups, part of whichare in the form of reactive esters, hydrazides, thiols, or reactivedisulfide-containing derivatives.
 10. The matrix coating according toclaim 1, wherein said charged groups and said reactive groups of saidhydrogel are selected from the group consisting of hydroxyl groups,carboxyl groups, amino groups, aldehyde groups, carbonyl groups, epoxygroups, and vinyl groups for immobilizing a desired ligand, and,optionally, a biospecific ligand bound via said groups.
 11. The matrixcoating according to claim 1, wherein said charged groups are carboxylgroups.
 12. The matrix coating according to claim 1, wherein thereactive groups are reactive esters, hydrazides or reactivedisulfide-containing derivatives.
 13. The matrix coating according toclaim 12, wherein the reactive disulfide-containing derivative is theproduct of a reaction between a reactive ester and 2-(2-pyridinyldithio)ethanamine.
 14. The matrix coating according to claim 12, wherein saidhydrogel contains a 2-aminoethanethiol derivative.
 15. A sensing elementsuitable for use in a biosensor, comprising:a substrate; and a matrixcoating comprising a hydrogel supported on said substrate via which adesired ligand can be bound, which hydrogel has been activated tocontain (i) charged groups for bringing about a concentration ofbiomolecules carrying an opposite charge to that of said charged groups,and (ii) reactive groups for covalently binding said biomoleculesconcentrated on said matrix coating.